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Hemopoietic development requires firm control of cell proliferation and differentiation. Although recent research has revealed conserved function of transcription factors and signaling pathways regulating lineage commitment in hemopoietic development in Drosophila melanogaster and vertebrates, little is known about hemopoietic cytokines among the invertebrate phyla. In the present study, we show that differentiation and growth of hemopoietic stem cells in vitro from an invertebrate, Pacifastacus leniusculus, require an endogenous cytokine-like factor, astakine, containing a prokineticin (PK) domain. Astakine induces a strong hematopoiesis response in live animals. An astakine homologue was also found in the shrimp, Penaeus monodon. So far, PK domains are only identified in vertebrates, in which they, for example, direct angiogenic growth. Our finding of the first PK-like cytokine characterized from any invertebrate provides novel information concerning the evolution of growth factors and blood cell development.

A 14 amino acid residues proline/arginine-rich antibacterial peptide designated as astacidin 2 was purified and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. Astacidin 2 has a broad range of antibacterial activity against both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 2 is RPRPNYRPRPIYRP with an amidated C-terminal and the molecular mass is 1838 Da determined by mass spectrometry. Furthermore, the cDNA of three different crustin antibacterial homologs were isolated from a crayfish hemocyte EST library. RT-PCR was used to analyze the expression of the genes coding for astacidin 2 and P. leniusculus crustins (Plcrustin) 1–3 after bacterial challenge. The expression of Plcrustin1 was upregulated in both hemocytes and hematopoietic tissue after challenge with Gram-negative Escherichia coli or Acinetobacter ssp. non pathogenic bacteria as well as by a Gram negative crayfish pathogenic bacterium (Aeromonas hydrophila). The PlCrustin3 transcript was only upregulated after inoculation with the non-pathogenic Acinetobacter ssp. while there was no change in expression of Plcrustin2 or astacidin 2 following a bacterial challenge.